Project Summary My laboratory has a long-standing interest in immune tolerance in transplantation and autoimmunity, with a focus on T cells and regulatory T cells (Tregs). One key pathway controlling T cell specification and responsiveness is the phosphoinositide 3-kinase (PI3K) pathway, which is activated via multiple surface receptors, including CD28 and the IL-2R. A central element downstream of the PI3K pathway is the transcription factor Foxo1. In quiescent cells, Foxo1 is restricted to the nucleus and maintains transcriptional activity; activation induces Foxo1 phosphorylation by Akt and resultant nuclear exclusion. While published studies have promoted a model in which Foxo1 nuclear expression is necessary and sufficient for normal T cell function, preliminary data from our laboratory suggest a much more complex picture. To address the role of Foxo1 transcriptional acitivity we have bred strains of mice that express an Akt- insensitive, constitutively active Foxo1 mutant (caFoxo1) specifically: (a) in all T cells, (b) inducibly by tamoxifen in CD4 T cells, and (c) in Foxp3+ Tregs. Using these mice we have made several observations: (1) Mice with dysregulated Foxo1 in all T cells have severe autoimmunity due to Treg dysfunction. However, when autoimmunity is prevented in mixed bone-marrow chimeras, or using the tamoxifen-inducible system, a specific defect in CD4 (but not CD8) T cells is revealed, consisting of hyperproliferation, loss of competitive fitness, and failure of Th1 differentiation. (2) In Tregs, we found that Treg-restricted expression of caFoxo1 results in a severe disruption of Treg stability and function in a gene dosage dependent manner. We propose a model in which, contrary to expectations, disruption of Foxo1 regulation does not result in an intrinsic loss of T cell quiescence, but rather in loss of homeostatic mechanisms that specifically maintain both CD4+ conventional and regulatory T cell fitness, without affecting CD8+ T cells in the steady state. In this application, we will study how modulation of Foxo1 transcriptional activity controls developing and mature T cells and Tregs. To do so, we have two aims. In Aim #1 we will use constitutive and inducible models of caFoxo1 expression, and studies of cytokine signaling and gene expression, to determine why Foxo1 transcriptional activity leads to selective loss of fitness in CD4 T cells. In Aim #2 we will focus on Tregs. Here, we will examine the direct effects of graded levels of caFoxo1 expression on Treg function, migration and stability by studying spontaneous autoimmunity, as well as induced disease models. These results will provide important insights into the mechanism and effects of current therapeutic strategies that target PI3K signaling, and to future strategies that may more specifically target Foxos in clinical settings that are dependent on differentially impacting effector T cells vs Tregs for their outcome.